Experimental Milestones in the Discovery of Molecular Chaperones as Polypeptide Unfolding Enzymes.

Molecular chaperones control the cellular folding, assembly, unfolding, disassembly, translocation, activation, inactivation, disaggregation, and degradation of proteins. In 1989, groundbreaking experiments demonstrated that a purified chaperone can bind and prevent the aggregation of artificially unfolded polypeptides and use ATP to dissociate and convert them into native proteins. A decade later, other chaperones were shown to use ATP hydrolysis to unfold and solubilize stable protein aggregates, leading to their native refolding. Presently, the main conserved chaperone families Hsp70, Hsp104, Hsp90, Hsp60, and small heat-shock proteins (sHsps) apparently act as unfolding nanomachines capable of converting functional alternatively folded or toxic misfolded polypeptides into harmless protease-degradable or biologically active native proteins. Being unfoldases, the chaperones can proofread three-dimensional protein structures and thus control protein quality in the cell. Understanding the mechanisms of the cellular unfoldases is central to the design of new therapies against aging, degenerative protein conformational diseases, and specific cancers.

The mRNA binding-mediated self-regulatory function of small heat shock protein IbpA in γ-proteobacteria is conferred by a conserved arginine.

Bacterial small heat shock proteins, such as inclusion body-associated protein A (IbpA) and IbpB, coaggregate with denatured proteins and recruit other chaperones for the processing of aggregates thereby assisting in protein refolding. In addition, as a recently revealed uncommon feature, Escherichia coli IbpA self-represses its own translation through interaction with the 5'-untranslated region of the ibpA mRNA, enabling IbpA to act as a mediator of negative feedback regulation. Although IbpA also suppresses the expression of IbpB, IbpB does not have this self-repression activity despite the two Ibps being highly homologous. In this study, we demonstrate that the self-repression function of IbpA is conserved in other γ-proteobacterial IbpAs. Moreover, we show a cationic residue-rich region in the α-crystallin domain of IbpA, which is not conserved in IbpB, is critical for the self-suppression activity. Notably, we found arginine 93 (R93) located within the α-crystallin domain is an essential residue that cannot be replaced by any of the other 19 amino acids including lysine. We observed that IbpA-R93 mutants completely lost the interaction with the 5' untranslated region of the ibpA mRNA, but retained almost all chaperone activity and were able to sequester denatured proteins. Taken together, we propose the conserved Arg93-mediated translational control of IbpA through RNA binding would be beneficial for a rapid and massive supply of the chaperone on demand.

The Chaperone Activity of the Developmental Small Heat Shock Protein Sip1 Is Regulated by pH-Dependent Conformational Changes.

Small heat shock proteins (sHsps) are ubiquitous molecular chaperones that prevent the aggregation of unfolding proteins during proteotoxic stress. In Caenorhabditis elegans, Sip1 is the only sHsp exclusively expressed in oocytes and embryos. Here, we demonstrate that Sip1 is essential for heat shock survival of reproducing adults and embryos. X-ray crystallography and electron microscopy revealed that Sip1 exists in a range of well-defined globular assemblies consisting of two half-spheres, each made of dimeric "spokes." Strikingly, the oligomeric distribution of Sip1 as well as its chaperone activity depend on pH, with a trend toward smaller species and higher activity at acidic conditions such as present in nematode eggs. The analysis of the interactome shows that Sip1 has a specific substrate spectrum including proteins that are essential for embryo development.

Role of Small Heat Shock Proteins in the Remodeling of Actin Microfilaments.

Small heat shock proteins (sHsps) play an important role in the maintenance of proteome stability and, particularly, in stabilization of the cytoskeleton and cell contractile apparatus. Cell exposure to different types of stress is accompanied by the translocation of sHsps onto actin filaments; therefore, it is commonly believed that the sHsps are true actin-binding proteins. Investigations of last years have shown that this assumption is incorrect. Stress-induced translocation of sHsp to actin filaments is not the result of direct interaction of these proteins with intact actin, but results from the chaperone-like activity of sHsps and their interaction with various actin-binding proteins. HspB1 and HspB5 interact with giant elastic proteins titin and filamin thus providing an integrity of the contractile apparatus and its proper localization in the cell. HspB6 binds to the universal adapter protein 14-3-3 and only indirectly affects the structure of actin filament. HspB7 interacts with filamin C and controls actin filament assembly. HspB8 forms tight complex with the universal regulatory and adapter protein Bag3 and participates in the chaperone-assisted selective autophagy (CASA) of actin-binding proteins (e.g., filamin), as well as in the actin-depending processes taking place in mitoses. Hence, the mechanisms of sHsp participation in the maintenance of the contractile apparatus and cytoskeleton are much more complicated and diverse than it has been postulated earlier and are not limited to direct interactions of sHsps with actin. The old hypothesis on the direct binding of sHsps to intact actin should be revised and further detailed investigation on the sHsp interaction with minor proteins participating in the formation and remodeling of actin filaments is required.

Functional analysis of a novel orthologous small heat shock protein (shsp) hsp21.8a and seven species-specific shsps in Tribolium castaneum.

Small heat shock proteins (sHSPs) are important modulators of insect survival. Previous research revealed that there is only one orthologous cluster of shsps in insects. Here, we identified another novel orthologous cluster of shsps in insects by comparative analysis. Multiple stress experiments and function investigation of Tchsp21.8a belonging to this orthologous cluster and seven species-specific shsps were performed in the stored-grain pest Tribolium castaneum. The results indicated that expression of Tchsp21.8a showed weak responses to different stresses. However, expressions of most species-specific shsps exhibited hyper-responses to heat stress, and expressions of all species-specific shsps displayed diverse responses during other stresses to protect beetles in a cooperative manner. Additionally, Tchsp21.8a and species-specific Tcshsp19.7 played important roles in the development of T. castaneum, and all Tcshsps had a certain impact on the fecundity. Our work created a comprehensive reliable scaffold of insect shsps that can further provide instructive insights to pest bio-control.

AgsA oligomer acts as a functional unit.

AgsA (aggregation-suppressing protein) is an ATP-independent molecular chaperone machine belonging to the family of small heat shock proteins (sHSP), and it can prevent the aggregation of non-natural proteins. However, the substrate-binding site of AgsA and the functional unit that captures and binds the substrate remain unknown. In this study, different N-terminal and C-terminal deletion mutants of AgsA were constructed and their effects on AgsA oligomer assembly and chaperone activity were investigated. We found that the IXI motif at the C-terminus and the α-helix at the N-terminus affected the oligomerization and molecular chaperone activity of AgsA. In this work, we obtained a 6.8 Å resolution structure of AgsA using Electron cryo-microscopy (cryo-EM), and found that the functional form of AgsA was an 18-mer with D3 symmetry. Through amino acid mutations, disulfide bonds were introduced into two oligomeric interfaces, namely dimeric interface and non-partner interface. Under oxidation and reduction conditions, the chaperone activity of the disulfide-bonded AgsA did not change significantly, indicating that AgsA would not dissociate to achieve chaperone activity. Therefore, we concluded that the oligomer, especially 18-mer, was the primary functional unit.

Identifications, Characteristics, and Expression Patterns of Small Heat Shock Protein Genes in a Major Mulberry Pest, Glyphodes pyloalis (Lepidoptera: Pyralidae).

Six candidate sHSP genes were identified from the Glyphodes pyloalis transcriptome. All sHSP genes included full-length open reading frames and shared high similarity with the sequences of other lepidopteran species. These sHSP genes encoded 175-191 amino acid residues, and the predicted proteins had a molecular weight from 19.5 to 21.8 kDa. All GpsHSPs were expressed at lower levels at larval stages. All GpsHSPs were expressed at higher levels at diapaused, prepupal, or pupal stages, suggesting that sHSPs may be involved in metamorphosis in G. pyloalis. In addition to the developmental stage, extreme temperatures can induce variations in the expression of sHSPs genes. All GpsHSPs were significantly upregulated in larvae following exposure to heat shock, except GpHSP21.4 which downregulated at 4 h following exposure to the cold shock treatment. Furthermore, Starvation influenced the expression patterns of GpsHSPs as a function of the duration of food deprivation. Four GpsHSPs increased their expression with time of starvation until reaching to the peak level at 6 d of starvation. Finally, parasitism by the endoparasitoid Aulacocentrum confusum He et van Achterberg (Hymenoptera: Braconidae)-induced fluctuations in the expression of all GpsHSPs, and the expression varied with time after parasitization. Our results from this study strongly suggest functional differentiation within the sHSPs subfamily in G. pyloalis. The present study would provide further insight into the roles of sHSPs in G. pyloalis and novel avenues for promoting integrated management of this pest.

The Heterooligomerization of Human Small Heat Shock Proteins Is Controlled by Conserved Motif Located in the N-Terminal Domain.

Ubiquitously expressed human small heat shock proteins (sHsps) HspB1, HspB5, HspB6 and HspB8 contain a conserved motif (S/G)RLFD in their N-terminal domain. For each of them, we prepared mutants with a replacement of the conserved R by A (R/A mutants) and a complete deletion of the pentapeptide (Δ mutants) and analyzed their heterooligomerization with other wild-type (WT) human sHsps. We found that WT HspB1 and HspB5 formed heterooligomers with HspB6 only upon heating. In contrast, both HspB1 mutants interacted with WT HspB6 even at low temperature. HspB1/HspB6 heterooligomers revealed a broad size distribution with equimolar ratio suggestive of heterodimers as building blocks, while HspB5/HspB6 heterooligomers had an approximate 2:1 ratio. In contrast, R/A or Δ mutants of HspB6, when mixed with either HspB1 or HspB5, resulted in heterooligomers with a highly variable molar ratio and a decreased HspB6 incorporation. No heterooligomerization of HspB8 or its mutants with either HspB1 or HspB5 could be detected. Finally, R/A or Δ mutations had no effect on heterooligomerization of HspB1 and HspB5 as analyzed by ion exchange chromatography. We conclude that the conserved N-terminal motif plays an important role in heterooligomer formation, as especially pronounced in HspB6 lacking the C-terminal IXI motif.

Dodecameric structure of a small heat shock protein from Mycobacterium marinum M.

Small heat shock proteins (sHSPs) are ATP-independent molecular chaperones present ubiquitously in all kingdoms of life. Their low molecular weight subunits associate to form higher order structures. Under conditions of stress, sHSPs prevent aggregation of substrate proteins by undergoing rapid changes in their conformation or stoichiometry. Polydispersity and dynamic nature of these proteins have made structural investigations through crystallography a daunting task. In pathogens like Mycobacteria, sHSPs are immuno-dominant antigens, enabling survival of the pathogen within the host and contributing to disease persistence. We characterized sHSPs from Mycobacterium marinum M and determined the crystal structure of one of these. The protein crystallized in three different conditions as dodecamers, with dimers arranged in a tetrahedral fashion to form a closed cage-like architecture. Interestingly, we found a pentapeptide bound to the dodecamers revealing one of the modes of sHSP-substrate interaction. Further, we have observed that ATP inhibits the chaperoning activity of the protein.

Chaperone-like activity of the N-terminal region of a human small heat shock protein and chaperone-functionalized nanoparticles.

Small heat shock proteins (sHsps) are molecular chaperones employed to interact with a diverse range of substrates as the first line of defense against cellular protein aggregation. The N-terminal region (NTR) is implicated in defining features of sHsps; notably in their ability to form dynamic and polydisperse oligomers, and chaperone activity. The physiological relevance of oligomerization and chemical-scale mode(s) of chaperone function remain undefined. We present novel chemical tools to investigate chaperone activity and substrate specificity of human HspB1 (B1NTR), through isolation of B1NTR and development of peptide-conjugated gold nanoparticles (AuNPs). We demonstrate that B1NTR exhibits chaperone capacity for some substrates, determined by anti-aggregation assays and size-exclusion chromatography. The importance of protein dynamics and multivalency on chaperone capacity was investigated using B1NTR-conjugated AuNPs, which exhibit concentration-dependent chaperone activity for some substrates. Our results implicate sHsp NTRs in chaperone activity, and demonstrate the therapeutic potential of sHsp-AuNPs in rescuing aberrant protein aggregation.

Sequence characteristics responsible for protein-protein interactions in the intrinsically disordered regions of caseins, amelogenins, and small heat-shock proteins.

Milk caseins and dental amelogenins are intrinsically disordered proteins (IDPs) that associate with themselves and others. Paradoxically, they are also described as hydrophobic proteins, which is difficult to reconcile with a solvent-exposed conformation. We attempt to resolve this paradox. We show that caseins and amelogenins are not hydrophobic proteins but they are more hydrophobic than most IDPs. Remarkably, uncharged residues from different regions of these mature proteins have a nearly constant average hydropathy but these regions exhibit different charged residue frequencies. A novel sequence analysis method was developed to identify hydrophobic and order-promoting regions that would favor conformational collapse. We found that such regions were uncommon; most hydrophobic and order-promoting residues were adjacent to hydrophilic or disorder-promoting residues. A further reason why caseins and amelogenins do not collapse is their high proportion of disorder-promoting proline residues. We conclude that in these proteins the hydrophobic effect is not large enough to cause conformational collapse but it can contribute, along with polar interactions, to protein-protein interactions. This behaviour is similar to the interaction of the disordered N-terminal region of small heat-shock proteins with either themselves during oligomer formation or other, unfolding, proteins during chaperone action.

Small Heat Shock Proteins and Human Neurodegenerative Diseases.

The review discusses the role of small heat shock proteins (sHsps) in human neurodegenerative disorders, such as Charcot-Marie-Tooth disease (CMT), Parkinson's and Alzheimer's diseases, and different forms of tauopathies. The effects of CMT-associated mutations in two small heat shock proteins (HspB1 and HspB8) on the protein stability, oligomeric structure, and chaperone-like activity are described. Mutations in HspB1 shift the equilibrium between different protein oligomeric forms, leading to the alterations in its chaperone-like activity and interaction with protein partners, which can induce damage of the cytoskeleton and neuronal death. Mutations in HspB8 affect its interaction with the adapter protein Bag3, as well as the process of autophagy, also resulting in neuronal death. The impact of sHsps on different forms of amyloidosis is discussed. Experimental studies have shown that sHsps interact with monomers or small oligomers of amyloidogenic proteins, stabilize their structure, prevent their aggregation, and/or promote their specific proteolytic degradation. This effect might be due to the interaction between the ß-strands of sHsps and ß-strands of target proteins, which prevents aggregation of the latter. In cooperation with the other heat shock proteins, sHsps can promote disassembly of oligomers formed by amyloidogenic proteins. Despite significant achievements, further investigations are required for understanding the role of sHsps in protection against various neurodegenerative diseases.

Comparative Membrane Proteomics Reveals a Nonannotated E. coli Heat Shock Protein.

Recent advances in proteomics and genomics have enabled discovery of thousands of previously nonannotated small open reading frames (smORFs) in genomes across evolutionary space. Furthermore, quantitative mass spectrometry has recently been applied to analysis of regulated smORF expression. However, bottom-up proteomics has remained relatively insensitive to membrane proteins, suggesting they may have been underdetected in previous studies. In this report, we add biochemical membrane protein enrichment to our previously developed label-free quantitative proteomics protocol, revealing a never-before-identified heat shock protein in Escherichia coli K12. This putative smORF-encoded heat shock protein, GndA, is likely to be ∼36-55 amino acids in length and contains a predicted transmembrane helix. We validate heat shock-regulated expression of the gndA smORF and demonstrate that a GndA-GFP fusion protein cofractionates with the cell membrane. Quantitative membrane proteomics therefore has the ability to reveal nonannotated small proteins that may play roles in bacterial stress responses.

Chaperone-client interactions between Hsp21 and client proteins monitored in solution by small angle X-ray scattering and captured by crosslinking mass spectrometry.

The small heat shock protein (sHsp) chaperones are important for stress survival, yet the molecular details of how they interact with client proteins are not understood. All sHsps share a folded middle domain to which is appended flexible N- and C-terminal regions varying in length and sequence between different sHsps which, in different ways for different sHsps, mediate recognition of client proteins. In plants there is a chloroplast-localized sHsp, Hsp21, and a structural model suggests that Hsp21 has a dodecameric arrangement with six N-terminal arms located on the outside of the dodecamer and six inwardly-facing. Here, we investigated the interactions between Hsp21 and thermosensitive model substrate client proteins in solution, by small-angle X-ray scattering (SAXS) and crosslinking mass spectrometry. The chaperone-client complexes were monitored and the Rg -values were found to increase continuously during 20 min at 45°, which could reflect binding of partially unfolded clients to the flexible N-terminal arms of the Hsp21 dodecamer. No such increase in Rg -values was observed with a mutational variant of Hsp21, which is mainly dimeric and has reduced chaperone activity. Crosslinking data suggest that the chaperone-client interactions involve the N-terminal region in Hsp21 and only certain parts in the client proteins. These parts are peripheral structural elements presumably the first to unfold under destabilizing conditions. We propose that the flexible and hydrophobic N-terminal arms of Hsp21 can trap and refold early-unfolding intermediates with or without dodecamer dissociation.

Large potentials of small heat shock proteins.

Modern classification of the family of human small heat shock proteins (the so-called HSPB) is presented, and the structure and properties of three members of this family are analyzed in detail. Ubiquitously expressed HSPB1 (HSP27) is involved in the control of protein folding and, when mutated, plays a significant role in the development of certain neurodegenerative disorders. HSPB1 directly or indirectly participates in the regulation of apoptosis, protects the cell against oxidative stress, and is involved in the regulation of the cytoskeleton. HSPB6 (HSP20) also possesses chaperone-like activity, is involved in regulation of smooth muscle contraction, has pronounced cardioprotective activity, and seems to participate in insulin-dependent regulation of muscle metabolism. HSPB8 (HSP22) prevents accumulation of aggregated proteins in the cell and participates in the regulation of proteolysis of unfolded proteins. HSPB8 also seems to be directly or indirectly involved in regulation of apoptosis and carcinogenesis, contributes to cardiac cell hypertrophy and survival and, when mutated, might be involved in development of neurodegenerative diseases. All small heat shock proteins play important "housekeeping" roles and regulate many vital processes; therefore, they are considered as attractive therapeutic targets.

Developmental Expression and Functions of the Small Heat Shock Proteins in Drosophila.

Heat shock proteins (Hsps) form a large family of evolutionarily conserved molecular chaperones that help balance protein folding and protect cells from various stress conditions. However, there is growing evidence that Hsps may also play an active role in developmental processes. Here, we take the example of developmental expression and function of one class of Hsps characterized by low molecular weight, the small Hsps (sHsps). We discuss recent reports and genome-wide datasets that support vital sHsps functions in the developing nervous system, reproductive system, and muscles. This tissue- and time-specific sHsp expression is developmentally regulated, so that the enhancer sequence of an sHsp gene expressed in developing muscle, in addition to stress-inducible elements, also carries binding sites for myogenic regulatory factors. One possible reason for sHsp genes to switch on during development and in non-stress conditions is to protect vital developing organs from environmental insults.

The Role of the Arginine in the Conserved N-Terminal Domain RLFDQxFG Motif of Human Small Heat Shock Proteins HspB1, HspB4, HspB5, HspB6, and HspB8.

Although the N-terminal domain of vertebrate small heat shock proteins (sHsp) is poorly conserved, it contains a core motif preserved in many members of the sHsp family. The role of this RLFDQxFG motif remains elusive. We analyzed the specific role of the first arginine residue of this conserved octet sequence in five human sHsps (HspB1, HspB4, HspB5, HspB6, and HspB8). Substitution of this arginine with an alanine induced changes in thermal stability and/or intrinsic fluorescence of the related HspB1 and HspB8, but yielded only modest changes in the same biophysical properties of HspB4, HspB5, and HspB6 which together belong to another clade of vertebrate sHsps. Removal of the positively charged Arg side chain resulted in destabilization of the large oligomers of HspB1 and formation of smaller size oligomers of HspB5. The mutation induced only minor changes in the structure of HspB4 and HspB6. In contrast, the mutation in HspB8 was accompanied by shifting the equilibrium from dimers towards the formation of larger oligomers. We conclude that the RLFDQxFG motif plays distinct roles in the structure of several sHsp orthologs. This role correlates with the evolutionary relationship of the respective sHsps, but ultimately, it reflects the sequence context of this motif.

Substrates of the chloroplast small heat shock proteins 22E/F point to thermolability as a regulative switch for heat acclimation in Chlamydomonas reinhardtii.

KEY MESSAGE: We have identified 39 proteins that interact directly or indirectly with high confidence with chloroplast HSP22E/F under heat stress thus revealing chloroplast processes affected by heat. Under conditions promoting protein unfolding, small heat shock proteins (sHsps) prevent the irreversible aggregation of unfolding proteins by integrating into forming aggregates. Aggregates containing sHsps facilitate the access of Hsp70 and ClpB/Hsp104 chaperones, which in ATP-dependent reactions disentangle individual proteins from the aggregates and assist in their refolding to the native state. Chlamydomonas reinhardtii encodes eight different sHsps (HSP22A to H). The goal of this work was to identify chloroplast-targeted sHsps in Chlamydomonas and to obtain a comprehensive list of the substrates with which they interact during heat stress in order to understand which chloroplast processes are disturbed under heat stress. We show that HSP22E and HSP22F are major chloroplast-targeted sHsps that have emerged from a recent gene duplication event resulting from the ongoing diversification of sHsps in the Volvocales. HSP22E/F strongly accumulate during heat stress and form high molecular mass complexes. Using differential immunoprecipitation, mass spectrometry and a stringent filtering algorithm we identified 39 proteins that with high-confidence interact directly or indirectly with HSP22E/F under heat stress. We propose that the apparent thermolability of several of these proteins might be a desired trait as part of a mechanism enabling Chlamydomonas chloroplasts to rapidly react to thermal stress.

Methylglyoxal and Small Heat Shock Proteins.

Methylglyoxal is a highly reactive dicarbonyl compound formed during glucose metabolism and able to modify phospholipids, nucleic acids, and proteins belonging to the so-called dicarbonyl proteome. Small heat shock proteins participating in protection of the cell against different unfavorable conditions can be modified by methylglyoxal. The probability of methylglyoxal modification is increased in the case of distortion of glucose metabolism (diabetes), in the case of utilization of glycolysis as the main source of energy (malignancy), and/or at low rate of modified protein turnover. We have analyzed data on modification of small heat shock protein HspB1 in different tumors and under distortion of carbohydrate metabolism. Data on the effect of methylglyoxal modification on stability, chaperone-like activity, and antiapoptotic activity of HspB1 were analyzed. We discuss data on methylglyoxal modifications of lens α-crystallins. The mutual dependence and mutual effects of methylglyoxal modification and other posttranslational modifications of lens crystallins are analyzed. We conclude that although there is no doubt that the small heat shock proteins undergo methylglyoxal modification, the physiological significance of this process remains enigmatic, and new experimental approaches should be developed for understanding how this type of modification affects functioning of small heat shock proteins in the cell.

[Recombinant small heat shock protein from Acholeplasma laidlawii increases the Escherichia coli viability in thermal stress by selective protein rescue].

In both prokaryotes and eukaryotes, the survival at temperatures considerably exceeding the optimum is supported by intense synthesis of the so-called heat shock proteins (HSPs), which act to overcome the adverse effects of heat stress. Among mycoplasmas (class Mollicutes), which have significantly reduced genomes, only some members of the Acholeplasmataceae family possess small HSPs of the α-crystallin type. Overproduction of a recombinant HSP IbpA (Hsp20) from the free-living mycoplasma Acholeplasma laidlawii was shown to increase the resistance of Escherichia coli to short-term heat shock. It has been long assumed that IbpA prevents protein aggregation and precipitation thereby increasing viability of E. coli cells. Several potential target proteins interacting with IbpA under heat stress were identified, including biosynthetic enzymes, enzymes of energy metabolism, and components of the protein synthesis machinery. Statistical analysis of physicochemical properties indicated that IbpA interaction partners significantly differ in molecular weight, charge, and isoelectric point from other members of the E. coli proteome. Upon shortterm exposure to increased temperature, IbpA was found to preferentially interact with high-molecular weight proteins having a pI of about 5.1, significantly lower than the typical values of E. coli proteins.